Rg1 antibodies and uses thereof

ABSTRACT

The present invention relates to antibodies, and antigen-binding antibody fragments, directed against an RG1 polypeptide. The invention further relates to methods for utilizing the antibodies, and antibody fragments, for diagnostic and therapeutic applications.

This application is a continuation of U.S. Ser. No. 11/981,219, filedOct. 30, 2007, now U.S. Pat. No. 7,611,706, which was a divisionalapplication of U.S. Ser. No. 10/895,183, filed Jul. 20, 2004, now U.S.Pat. No. 7,335,748, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/489,032, filed on Jul. 22, 2003, the contents ofall of which are herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to novel antibodies directed against apolypeptide, RG1, which is preferentially expressed in prostate andother tumor cells. In particular, the invention relates to the use ofthese antibodies for the treatment and detection of cancer and cancermetastasis.

BACKGROUND OF THE INVENTION

Prostate cancer is a frequently occurring disease in man, in that it isfound in about one third of men over the age of 45. There is evidencefor both genetic and environmental causes, with the majority of casesprobably being the result of a combination of both factors. Studies offamilial cancer have suggested that genetic predisposition plays a rolein about 5-10% of all prostate cancers, and in about 45% of cases in menyounger than 55.

There is evidence that prostate cancer develops as a multi-step disease,with one of the precursor lesions being prostatic intraepithelialneoplasia (PIN). Early stages of the disease are androgen dependent,while later stages are hormone independent. A proliferative disorder ofthe prostate known as benign prostatic hyperplasia is often detectedclinically but is probably not a stage in the development of cancer. Itis, however, frequently associated with prostate cancer. Cancers in theprostate are often multifocal, generally slow growing, andheterogeneous. Late stage cancers frequently metastasize to the lymphnodes and to the bone.

Prostate cancer is usually diagnosed by physical examination and byserum levels of prostate specific antigen (PSA). Radical prostatectomyis the treatment of choice for localized disease. Advanced metastaticdisease is treated currently by androgen ablation induced by orchiectomyor treatment with GnRH (gonadotrophin releasing hormone), and byanti-androgen therapy. However, advanced disease almost invariablybecomes hormone resistant and there is no cure for progressive disease.Moreover, there are serious side effects associated with both radicalprostatectomy and androgen ablation therapy. These include a high riskof incontinence and impotence associated with radical prostatectomy andbone fractures and osteoporosis associated with androgen ablationtherapy.

There is, therefore, a considerable-need for new therapeutic approachesfor both early and late stage prostate cancer. There is also asignificant need for new diagnostic agents, as this significantlyinfluences the treatment options. For example, if disease has progressedbeyond the prostate and has metastasized to the lymph nodes, radicalprostatectomy is not undertaken as it has no effect on progression, butmay have significant unwanted side effects. An agent that could detectmetastasis, in vivo, would have considerable value.

Changes in the expression of specific proteins have been demonstrated inprostate cancer including abnormal p53 expression in late stage prostatecancer, reduced levels of TGF-3 receptors, reduced levels of E-cadherin,C-Cam (a cell adhesion molecule), and several integrins. The expressionof the oncogene bcl-2 is strikingly elevated in late stage androgenindependent tumors, and prognosis for patients expression bcl-2 atelevated levels is relatively poor. While the previously mentionedchanges in gene expression are well documented, no changes in expressionhave been identified that have been demonstrated to be causative for thedisease. It would, therefore, be useful to identify new proteins whoseexpression is linked to the presence or development of prostate tumorsthat could serve as molecular targets for compositions directed toprostate cancer diagnosis and therapy.

The polypeptide RG1 (see U.S. Pat. No. 5,871,969) is a homolog of theMindin/F-spondin family, which are extracellular matrix proteins. TheRG1 polypeptide was demonstrated to be highly expressed in prostatetissue (see WO98/45442), and should be a useful target for diagnosis andtherapy of prostate cancer, as well as in other cancers where it isexpressed.

SUMMARY OF THE INVENTION

The present invention provides antibodies, or antigen-binding antibodyfragments thereof, or variants thereof, that are highly selective forRG1 polypeptides, and which may be employed in methods for detection ofRG1 expression, which is associated with disease states such as cancerof the prostate, kidney, colon or ovaries, and in the treatment of suchdisease states.

Toward these ends, it is an object of the invention to provide isolatedantibodies, or antigen-binding antibody fragments thereof, or variantsthereof, that specifically bind to an epitope present in an RG1polypeptide (SEQ ID NO: 2). Particularly preferred are human antibodiesthat bind to epitope of the RG1 polypeptide with a dissociation constant(K_(D)) which is less than or equal to 1 μM, more preferably less thanor equal to 100 nM and most preferably less than or equal to 10 nM.

In accordance with further preferred embodiments of the invention,isolated antibodies and antigen-binding antibody fragments thereof,comprising a light chain variable region comprising the amino acidsequences of SEQ ID NO: 26 or SEQ ID NO: 29 are provided.

Also provided are isolated antibodies and antigen-binding antibodyfragments thereof, comprising a heavy chain variable region comprisingthe amino acid sequences SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 orSEQ ID NO: 31. A particularly preferred embodiment is a human antibody,comprising a light chain variable region having the amino acid sequenceSEQ ID NO: 26 and a heavy chain variable region having the amino acidsequence SEQ ID NO: 27 or SEQ ID NO: 28, A second particularly preferredembodiment is a human antibody, comprising a light chain variable regionhaving the amino acid sequence SEQ ID NO: 29 and a heavy chain variableregion having the amino acid sequence SEQ ID NO: 30 or SEQ ID NO: 31.

In a further aspect of the invention, light chain variable regions andheavy chain variable regions with amino acid sequences having 80%sequence identity to the amino acid sequences described above are alsocontemplated.

Also provided are nucleotide sequences which encode the light chain andheavy chain variable regions of the antibodies described above.Preferred is an antibody comprising a light chain variable regionencoded by a nucleotide sequence comprising SEQ ID NO: 20 or SEQ ID NO:23. Also preferred is an antibody comprising a heavy chain variableregion encoded by a nucleotide sequence comprising SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 24, or SEQ ID NO: 25.

In accordance with certain preferred embodiments of this aspect of theinvention, the antibodies are conjugated to a detectable marker, for useas a diagnostic agent for administration to cells in vitro, to cells exvivo and to cells in vivo, or to a multicellular organism. Particularlypreferred would be an antibody conjugated to a radiolabel, an enzyme, achromophore or a fluorescer. Particularly preferred methods of detectionare immunoscintigrapy and positron emitting tomography, in which theantibody would be conjugated to a radioiotope such as ¹¹¹In or ^(99m)Tc,for immunoscintigraphy, or to ⁴³Sc, ⁴⁴Sc, ⁵²Fe, ⁵⁵Co, ⁶⁶Ga, ⁶⁴Cu, ⁸⁶Y or^(94m)Tc, for positron emitting tomography.

In a further aspect of the invention there are provided antibodies thatare conjugated to a therapeutic agent, e.g. ricin or a radioisotope, foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. Preferred in this regard are therapeuticagents that are cytotoxic. Particularly preferred for a therapeuticagent would be antibodies conjugated to a radioisotope, such as ⁹⁰Y and¹⁷⁷Lu. In certain preferred embodiments in this regard is administrationof such conjugated antibodies to a human patient for treatment of adisease state characterized by RG1 expression, such as prostate cancer,and in particular, advanced metastatic prostate cancer.

In a further aspect of the invention, conjugation of an RG1 antibody, orantigen-binding fragment thereof, to a detectable marker or cytotoxicagent is accomplished through use of a chelator selected from the groupconsisting of p-SCN-Benzyl-DTPA and derivatives thereof,1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetracetic acid (DOTA) andderivatives thereof, and 1,4,7-triazacyclononane-N,N′,N″-triacetic acid(NOTA) and derivatives thereof.

In a further aspect of the invention is a method for treatment of adisease-state associated with expression of an RG1 polypeptide, such asprostate, which uses the immunoconjugates of the present invention.

In a further aspect of the invention is a method for detection of adisease-state associated with expression of an RG1 polypeptide, such asprostate cancer, which uses the immunoconjugates of the presentinvention.

In a further aspect of the invention, peptides and anti-idiotypicantibodies are provided which can be used to stimulate an immuneresponse.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Biodistribution of ¹¹¹In labeled RG1 Antibodies: Three RG1antibodies (A, B, and C), a non-specific hIgG, control antibody, and theantibody CYT-356 (PROSTASCINT™, Cytogen Corporation, Princeton, N.J.)were radiolabeled with ¹¹¹In. Radiolabeled antibodies (specificactivity: 0:3 mCi/mg) were administered i.v. into tumor (LNCaP) bearingnude mice. Twelve animals per group (3 animals per time point) weresacrificed at 6, 24, 120, and 150 hr p.i. and the accumulation in thetumor, blood and liver monitored. (See Example 11)

FIG. 2: Anti-tumor effect of ⁹⁰Y labeled RG1 Antibodies: LNCaP tumorbearing animals were injected with ⁹⁰Y labeled antibodies (anti-RG1antibodies B and C or non-specific IgG₁, specific activity, 0.5 mCi/mg).A single dose of 125 μCi ⁹⁰Y-labeled RG1 antibody (B,C) was administeredi.p. Mice were sacrificed at day 32 and tumors excised and weighed. (SeeExample 12)

FIG. 3: Amino acid sequence of the variable chain regions of humanmonoclonal antibody B, including a mutated variable heavy chain region.V_(L) (SEQ ID NO: 26), V_(H) (SEQ ID NO: 27), B_(—)3M, V_(H) (SEQ ID NO:28).

FIG. 4: Amino acid sequence of the variable chain regions of humanmonoclonal antibody C, including a mutated variable heavy chain region.V_(L) (SEQ ID NO: 29), V_(H) (SEQ ID NO: 30), C_(—)2m, V_(H) (SEQ ID NO:31).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in the specification, examples and appended claims, unlessspecified to the contrary, the following terms have the meaningindicated.

“rg1” refers to the polynucleotide having the sequence set out in SEQ IDNO: 1 and polynucleotides encoding polypeptides having the amino acidsequence of RG1 set out in SEQ ID NO: 2; and to polynucleotides encodingRG1 variants, derivatives and fragments, and fragments of the variantsand derivatives. Rg1 also refers to such polynucleotides composed of RNAas well as to polynucleotides which are the complement ofpolynucleotides which encode the polypeptide sequence set out in SEQ IDNO: 2.

“RG1” refers to tile polypeptide having the amino acid sequence set outin SEQ ID NO: 2, variants and derivatives thereof, and fragments of SEQID NO: 2, variants and derivatives thereof. The terms “variant”,“fragment” and “derivative”, when referring to the polypeptide of SEQ IDNO: 2 mean a polypeptide which retains essentially the same biologicand/or immunologic activity as the polypeptide of SEQ ID NO: 2.

“Biologic activity” refers to the structural, regulatory or biochemicalfunctions of naturally occurring RG1 polypeptide.

“Immunologic activity” refers to (1) the capability of the natural,recombinant or synthetic RG1, or any fragment thereof, to induce aspecific immune response in appropriate animals or cells and to bindwith specific antibodies, or (2) the capacity of antibodies to RG1 tobind RG1 in vivo and trigger an enhanced, cellular immune response toRG1 expressing tissue or tumor.

“Naturally occurring RG1” refers to RG1 produced by human cells thathave not been genetically engineered and specifically contemplatesvarious RG1 forms arising from post-translational modifications of thepolypeptide including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation, acylation, and cleavage.

“Native RG1” or “nRG1” refers to RG1 which is in its nativeconformation.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, polynucleotide as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term “polynucleotide” includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritium-labelled bases, to name just two examples, arepolynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term “polynucleotide” as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia.

“Oligonucleotide(s)” refers to relatively short polynucleotides. Oftenthe term refers to single-stranded deoxyribonucleotides, but it canrefer as well to single- or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others. Oligonucleotides, suchas single-stranded DNA probe oligonucleotides, often are synthesized bychemical methods, such as those implemented on automated oligonucleotidesynthesizers. However, oligonucleotides can be made by a variety ofother methods, including in vitro recombinant DNA-mediated techniquesand by expression of DNAs in cells and organisms. “Oligonucleotides” or“oligomers” or polynucleotide “fragment”, “portion”, or “segment” refersto a polynucleotide sequence of at least about 10 nucleotides and asmany as about 60 nucleotides, preferably about 15 to 30 nucleotides, andmore preferably about 20-25 nucleotides.

“Polypeptides”, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids at commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Common modifications include glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, and these and others are describedin most basic texts, such as, for instance, I. E. Creighton,Proteins-Structure and Molecular Properties, 2nd Ed., W.H. Freeman andCompany, New York, 1993. Many detailed reviews are available on thissubject, such as, for example, those provided by Wold, F., inPosttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pp 1-12, 1983; Seifter et al., Meth. Enzymol.182: 626-646, 1990 and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62, 1992.

It will be appreciated, as is well known, and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationalevents, including natural processing events and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationalnatural processes and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made, For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

“Derivative” refers to polynucleotides or polypeptides derived fromnaturally occurring rg1, RG1, or from antibodies binding RG1,respectively, by chemical modifications such as ubiquitination, labeling(e.g., with radionuclides, various enzymatic modifications), pegylation(derivatization with polyethylene glycol) or by insertion orsubstitution of amino acids such as ornithine (or substitution of thenucleotides which code for such as an amino acid), which do not normallyoccur in human proteins.

“Polynucleotide encoding a polypeptide” as used herein encompassespolynucleotides which include a sequence encoding a polypeptide of thepresent invention, particularly the RG1 polypeptide having the aminoacid sequence set out in SEQ ID NO: 2. The term encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the polypeptide (for example, interrupted by introns)together with additional regions.

A polypeptide “fragment”, “portion”, or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and invarious embodiments, at least about 17 or more amino acids. “Fragment”refers to a polypeptide having an amino acid sequence that is entirelythe same as part, but not all, of the amino acid sequence of theaforementioned RG1 polypeptides, or antibodies to RG1, and variants orderivatives thereof.

“Deletion” is defined as a change in either polynucleotide or amino acidsequences in which one or more polynucleotides or amino acid residues,respectively, are absent.

“Insertion” or “addition” is that change in a polynucleotide or aminoacid sequence which has resulted in the addition of one or morepolynucleotides or amino acid residues, respectively, as compared to thenaturally occurring polynucleotide or amino acid sequence.

“Substitution” results from the replacement of one or morepolynucleotides or amino acids by different polynucleotides or aminoacids, respectively.

“Variant(s)” of polynucleotides or polypeptides, as the term is usedherein, are described below and elsewhere in the present disclosure ingreater detail.

A variant of a polynucleotide is a polynucleotide that differs inpolynucleotide sequence from another, reference polynucleotide.Generally, differences are limited so that the polynucleotide sequencesof the reference and the variant are closely similar overall and, inmany regions, identical.

Changes in the polynucleotide sequence of the variant may be silent.That is, they may not 35 alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thepolynucleotide sequence of the variant may alter the amino acid sequenceof a polypeptide encoded by the reference polynucleotide. Suchpolynucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in tile polypeptideencoded by the reference sequence, as discussed below.

A variant of a polypeptide is a polypeptide that differs in amino acidsequence from another, reference polypeptide. Generally, differences arelimited so that the sequences of the reference and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination. Recombinant variants encoding these sameor similar polypeptides may be synthesized or selected by making use ofthe “redundancy” In the genetic code. Various codon substitutions, suchas the silent changes that produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsmay also be introduced to modify the properties of the polypeptide, tochange ligand-binding affinities, interchain affinities, or polypeptidedegradation or turnover rate.

As discussed herein, minor variations in the amino acid sequences ofpolypeptides, antibodies or immunoglobulin molecules are contemplated asbeing encompassed by the present invention, providing that thevariations in the amino acid sequence maintain at least 80%, morepreferably at least 85%, 90% 95%, and most preferably 99% of theoriginal sequence. In particular, conservative amino acid replacementsare contemplated. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into families: (1)acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine);(3) non-polar (alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan); and (4) uncharged polar(glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine).More preferred families are: serine and threonine are analiphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by comparing the specificactivity of the polypeptide derivative with the unmodified polypeptide.For purposes of this application, the invention encompasses variants ofthe claimed antibodies which maintain a binding affinity (K_(D)) lessthan 1 μM for an RG1 epitope.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, “substantial identity”, “similarity”, and“homologous”. A “reference sequence” is a defined sequence used as abasis for a sequence comparison; a reference sequence may be a subset ofa larger sequence, for example, as a segment of a full-length cDNA orgene sequence given in a sequence listing or may comprise a completecDNA or gene sequence. Generally, a reference sequence is at least 18nucleotides or 6 amino acids in length, frequently at least 24.nucleotides or 8 amino acids in length, and often at least 48nucleotides or 16 amino acids in length. Since two polynucleotides oramino acid sequences may each (1) comprise a sequence (i.e., a portionof the complete polynucleotide or amino acid sequence) that is similarbetween the two molecules, and (2) may further comprise a sequence thatis divergent between the two polynucleotides or amino acid sequences,sequence comparisons between two (or more) molecules are typicallyperformed by comparing sequences of the two molecules over a “comparisonwindow” to identify and compare local regions of sequence similarity. A“comparison window”, as used herein, refers to a conceptual segment ofat least 18 contiguous nucleotide positions or 6 amino acids wherein apolynucleotide sequence or amino acid sequence may be compared to areference sequence of at least 18 contiguous nucleotides or 6 amino acidsequences and wherein the portion of the polynucleotide sequence in thecomparison window may comprise additions, deletions, substitutions, andthe like (i.e., gaps) of 20 percent or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, (Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector softwarepackages), or by inspection, and the best alignment (i.e., resulting inthe highest percentage of homology over the comparison window) generatedby the various methods is selected,

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence. The term “similarity”, when used to describe apolypeptide, is determined by comparing the amino acid sequence and theconserved amino acid substitutes of one polypeptide to the sequence of asecond polypeptide. The term “homologous”, when used to describe apolynucleotide, indicates that two polynucleotides, or designatedsequences thereof, when optimally aligned and compared, are identical,with appropriate nucleotide insertions or deletions, in at least 70% ofthe nucleotides, usually from about 75% to 99%, and more preferably atleast about 98 to 99% of the nucleotides.

“Antibody” or “antigen-binding antibody fragment” refers to an intactantibody, or a fragment thereof, that competes with the intact antibodyfor specific binding. An antibody or antigen-binding antibody fragment,is said to specifically bind an antigen when the dissociation constantis less than or equal to 1 μM, preferably less than or equal to 100 nMand most preferably less than or equal to 10 nM, Binding can be measuredby methods known to those skilled in the art, an example being the useof a BIAcore™ instrument. Antibody fragments comprise a portion of anintact antibody, preferably the antigen binding or variable region ofthe intact antibody. Binding fragments include Fab, Fab′, F(ab′) and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments(C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughsin Molecular Biology), Oxford University Press; R. Kontermann & S,Duebel, editors (2001) Antibody Engineering (Springer LaboratoryManual), Springer Verlag). An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical.

“Epitope” includes any protein determinant capable of specific bindingto an immunoglobulin or T-cell receptor. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Two antibodies are said to “bind the same epitope” ifone antibody is shown to compete with the second antibody in acompetitive binding assay, by any of the methods well known to those ofskill in the art.

“Recombinant” or “recombinant DNA molecule” refers to a polynucleotidesequence which is not naturally occurring, or is made by the artificialcombination of two otherwise separated segments of sequence. By“recombinantly produced” is meant artificial combination oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of polynucleotides, e.g., by geneticengineering techniques. Such manipulation is usually done to replace acodon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a sequence recognitionsite. Alternatively, it is performed to join together polynucleotidesegments with desired functions to generate a single genetic entitycomprising a desired combination of functions not found in the commonnatural forms. Restriction enzyme recognition sites, regulationsequences, control sequences, or other useful features may beincorporated by design. “Recombinant DNA molecules” include cloning andexpression vectors. “Recombinant” may also refer to a polynucleotidewhich encodes a polypeptide and is prepared using recombinant DNAtechniques.

“Isolated” means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living animal inits natural state is not “isolated”, but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. For example, with respectto polynucleotides, the term isolated means that it is separated fromthe chromosome and cell in which it naturally occurs. Polynucleotidesand polypeptides may occur in a composition, such as media formulations,solutions for introduction of polynucleotides or polypeptides, forexample, into cells, compositions or solutions for chemical or enzymaticreactions, for instance, which are not naturally occurring compositions,and, therein remain isolated polynucleotides or polypeptides within themeaning of that term as it is employed herein.

“Substantially pure” and “substantially homogenous” are usedinterchangeably and describe RG1 polypeptide, or fragments thereof, or apolynucleotide segment encoding same, where such polypeptide orpolynucleotide is separated from components that naturally accompany it.An RG1 polypeptide or fragment thereof, or DNA segment encoding same issubstantially free of naturally-associated components when it isseparated from the native contaminants which accompany it in its naturalstate. Thus, a polypeptide that is chemically synthesized or synthesizedin a cellular system different from the cell in which it naturallyoriginates will be substantially free from its naturally-associatedcomponents. Similarly, a polynucleotide that is chemically synthesizedor synthesized in a cellular system different from the cell in which itnaturally originated will be substantially free from itsnaturally-associated components.

“Polymerase chain reaction” or “PCR” refers to a procedure whereinspecific pieces of DNA are amplified as described in U.S. Pat. No.4,683,195, issued 28 Jul. 1987. Generally, sequence information from theends of the polypeptide fragment of interest or beyond needs to beavailable, such that oligonucleotide primers can be designed; theseprimers will point towards one another, and will be identical or similarin sequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers will coincide with the ends ofthe amplified material. PCR can be used to amplify specific DNAsequences from total genomic DNA, cDNA transcribed from total cellularRNA, plasmid sequences, etc. (See generally Mullis et al., Cold SpringHarbor Symp. Quant Biol., 51: 263, 1987; Erlich, ed., PCR Technology,Stockton Press, NY, 1989).

“Stringency” typically occurs in a range from about T_(m), (meltingtemperature)-5° C. (5° below the T_(m) of the probe) to about 20° C. to25° C. below T_(m). As will be understood by those of skill in the art,a stringent hybridization can be used to identify or detect identicalpolynucleotide sequences or to identify or detect similar or relatedpolynucleotide sequences. As herein used, the term “stringentconditions” means hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences.

“Hybridization” as used herein, shall include “any process by which apolynucleotide strand joins with a complementary strand through basepairing” (Coombs, J., Dictionary of Biotechnology, Stockton Press, NewYork, N.Y., 1994).

“Therapeutically effective dose” refers to that amount of polypeptide orits antibodies, antagonists, or inhibitors, including antisensemolecules and ribozymes, which ameliorate the symptoms or conditions ofa disease state. A dose is considered a therapeutically effective dosein the treatment of cancer or its metastasis when tumor or metastaticgrowth is slowed or stopped, or the tumor or metastasis is found toshrink in size, so as to lead to an extension in life span for thesubject. A dose is also considered therapeutically effective if it leadsto an improvement in the overall quality of life of the patient, i.e.alleviation of pain. Therapeutic efficacy and toxicity of such compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED₅₀ (the dose therapeutically effectivein 50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀.

“Treating” or “treatment” as used herein covers the treatment of adisease-state in a human patient, which disease-state includes diseasestates which are characterized by an increased level of RG1, such asprostate cancer or advanced metastatic prostate cancer.

DETAILED DESCRIPTION OF THE INVENTION Antibodies

The present invention relates to antibodies, antigen-binding antibodyfragments thereof, and variants of the antibodies and fragments, thatspecifically bind to an RGI polypeptide, particularly to the RG1polypeptide having the amino acid sequence of SEQ ID NO; 2. Theseantibodies can be, for example, polyclonal or monoclonal antibodies.More preferred are monoclonal antibodies. Still more preferred arechimeric or humanized antibodies, and still more preferred are humanantibodies.

The antibodies, antigen-binding antibody fragments, and variants of theantibodies and fragments, contemplated in the present invention bind toan epitope of the RGI polypeptide with a dissociation constant (K_(D))less than or equal to 1 μM. More preferred are antibodies which bindwith a K_(D) less than or equal to 100 nM, Most preferred are antibodieswhich bind with a K_(D) less than or equal to 10 nM. Also contemplatedare antibodies which recognize and bind to the same epitope as theepitope bound by the antibodies described below, and which can bedetermined through competitive binding studies, using techniqueswell-known to those skilled in the art.

The antibodies, antigen-binding antibody fragments, and variants of theantibodies and fragments of the invention are comprised of a light chainvariable region and a heavy chain variable region. Among the preferredembodiments of the invention in this regard are antibodies,antigen-binding antibody fragments thereof, or variants thereof,comprising a light chain variable region having at least 80%, morepreferrably at least 90%, still more preferrably at least 95%, and stillmore preferrably 99% sequence identity to the amino acid sequences ofSEQ ID NO: 26 or SEQ ID NO: 29. Also preferred embodiments areantibodies, antigen-binding antibody fragments thereof, or variantsthereof, comprising a heavy chain variable region having at least 80%,more preferrably at least 90%, still more preferrably at least 95%, andstill more preferrably 99% sequence identity to the amino acid sequencesof SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30 or SEQ ID NO: 31 (seeFIGS. 3 and 4).

Particularly preferred embodiments of the invention are antibodies orantigen-binding antibody fragments thereof, or variants thereof,comprising a light chain variable region having the amino acid sequenceSEQ ID NO: 26 or SEQ ID NO: 29, which are encoded by the nucleotidesequences SEQ ID NOS: 20 and 23, respectively.

Also particularly preferred are antibodies, or antigen-binding antibodyfragments thereof, or variants thereof, comprising a heavy chainvariable region having an amino acid sequence chosen from SEQ ID NOS:27, 28, 30 or 31, which are encoded by the nucleotide sequences SEQ IDNOS. 21, 22, 24 and 25, respectively.

More particularly preferred in this regard are an antibody, orantigen-binding antibody fragment thereof, or a variant thereof,comprising a light chain variable region having the amino acid sequenceSEQ ID NO: 26 and further comprising a heavy chain variable regionhaving the amino acid sequence SEQ ID NO:27 or SEQ ID NO: 28 and asecond antibody comprising a light chain variable region having theamino acid sequence SEQ ID NO: 29 and further comprising a heavy chainvariable region having the amino acid sequence SEQ ID NO: 30 or SEQ IDNO: 31.

Most preferred are the human antibodies, or antigen-binding antibodyfragments thereof, or variants thereof, as follows: (a) an antibodycomprised of a light chain variable region having the amino acidsequence SEQ ID NO: 26 and a heavy chain variable region having theamino acid sequence SEQ ID NO: 27, (b) an antibody comprised of a lightchain variable region having the amino acid sequence SEQ ID NO: 26 and aheavy chain variable region having the amino acid sequence or SEQ ID NO:28, (c) an antibody comprised of a light chain variable region havingthe amino acid sequence SEQ ID NO: 29 and a heavy chain variable regionhaving the amino acid sequence SEQ ID NO: 30, or (d) an antibodycomprised of a light chain variable region having the amino acidsequence SEQ ID NO: 29 and a heavy chain variable region having theamino acid sequence or SEQ ID NO: 31.

Antibody Production

RG1 polypeptides, fragments or derivatives, or cells expressing them canbe used as an immunogen to produce antibodies thereto (Harlow,Antibodies, Cold Spring Harbor Press, NY (1989)). Various proceduresknown in the art may be used for the production of such antibodies andfragments (C. A. K Borrebaeck, editor (1995) Antibody Engineering(Breakthroughs in Molecular Biology), Oxford University Press; R.Kontermann & S. Duebel, editors (2001) Antibody Engineering (SpringerLaboratory Manual), Springer Verlag).

Antibodies generated against RG1 can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.Alternate methods that do not require the use of purified RG1 protein orRG1 peptides to generate antibodies to RG1, include ‘DNA immunization’in which an expression vector or virus is created using DNA coding forRG1 and is used to transfect or infect host tissue cells to express RG1in the animal used to generate antibodies, or cell based immunization inwhich cell lines expressing RG1 created in vitro are used in theimmunization procedure.

Monoclonal antibodies can be prepared using any technique which providesantibodies produced by continuous cell line cultures. Examples includethe hybridoma technique (Kohler and Milstein, Nature 256: 495-497,1975), the human B-cell hybridoma technique (Kozbor et al., ImmunologyToday 4: 72, 1983) and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al in Monoclonal Antibodies and Cancer,Alan R. Liss, Inc., 77-96, 1985). For cell based immunizations usingcell lines expressing RG1, subtractive immunization may be used toimmunotolerize the animals to the parent cell line (Sleister, H. M. andRao, A. G., J. Immunological Methods 261:213-220, 2002).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al., Proc. Natl. Acad. Sci.USA 81:6851-6855, 1984; Neuberger et al., Nature 312:604-608, 1984;Takeda et al., Nature 314:452-454, 1985). Alternatively, techniquesdescribed for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce RG1-specific single chainantibodies.

Furthermore, “human” antibodies can be produced using the methodsdescribed in U.S. Pat. Nos. 5,877,397 and 5,569,825, which areincorporated herein in full by reference, or through use of theXENOMOUSE™, as described in Mendez et al. Nature Genetics 15:146-156,1997. Such antibodies can also be generated using phage displaytechnology (Rader et al., Current Opinion in Biotechnology 8:155-I 168,1997; Aujame et al., Human Antibodies 8:155-168, 1997). The generationof human antibodies is very attractive, in that such antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomouse or mouse-derived monoclonal antibodies. Generation of humanantibodies which recognize epitopes of the RG1 polypeptide (SEQ ID NO:2) are described In Example 4.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (Proc. Natl. Acad. Sci. USA 86:3833-3837, 1989) andWinter and Milstein (Nature 349:293-299, 1991).

Antibody fragments which contain specific binding sites for RG1 may alsobe generated. There are often advantages to using antibody fragments,rather than whole antibodies, since the smaller size of the fragmentscan lead to more rapid clearance, and may also provide improved accessto solid tumors.

Such fragments include, but are not limited to the F(ab′)₂ fragmentswhich can be produced by pepsin digestion of the antibody molecule andthe Fab fragments which can be generated by reducing the disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse et al.,Science 256:1270-1281, 1989). Fab, Fv and ScFv antibody fragments canall be expressed in and secreted from E. coli, or a variety of eukaryotecell expression systems, allowing for the production of large amounts ofthese fragments. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). Other techniques forthe production of antibody fragments are known to those skilled in theart. Single chain Fv fragments (scFv), diabodies, minibodies and otherengineered antibody fragments are also envisioned (see U.S. Pat. Nos.5,5761,894 and 5,587,458; Hudson et al. Nature Medicine 9:129-133,2003)). Fv and sFv fragments are examples of species with intactcombining sites that are devoid of constant regions; thus, they arelikely to show reduced nonspecific binding during in vivo use, and areparticularly preferred for use as imaging agents (C. A. K Borrebaeck,editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology),Oxford University Press; R. Kontermann & S. Duebel, editors (2001)Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Theantibody fragment may also be a “linear antibody” e.g., as described inU.S. Pat. No. 5,641,870, for example. Such linear antibody fragments maybe monospecific or bispecific.

Variants of the antibodies or antibody fragments described herein arealso contemplated, and can be made using any of the techniques andguidelines for conservative and non-conservative mutations, e.g., U.S.Pat. No. 5,364,934. Variations include substitution, deletion orinsertion of one or more codons encoding the antibody, resulting in achange in the amino acid sequence as compared with the native antibodysequence. The utility of such variations contemplated would includethose leading to (1) a reduction in susceptibility to proteolysis orinactivation by oxidation, (2) an alteration in binding affinity forforming protein complexes or binding affinities to antigens, (3) analteration in in vivo clearance or biodistribution, (4) changes in theantibody isotype or allotype, (5) changes in the functional propertiesof the antibody, for example Fc receptor binding, (6) an alteration inthe epitope sequences to decrease or increase immunogenicity, and (7)other changes in physiocochemical or functional properties of suchanalogs. Guidance in determining which amino acid residue my beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by minimizing the number of amino acid sequencechanges made in regions of high homology between the RG1 antibodies andthat of homologous proteins. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activityexhibited by the native sequence.

A particularly preferred type of substitutional variant involvessubstituting one more hypervariable region residues of a parent antibody(e.g. human antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display (Schier R., J. Mol. Biol., 263:551-67, 1996). Thevariants are then screened for their biological activity (e.g. bindingaffinity) as described herein (see Example 4). In order to identifyhypervariable region residues which would be good candidates formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Antibodies with superior properties in one or more relevantassays can undergo further development.

The amino acid sequence of RG1 (SEQ ID NO: 2) presented herein may beused to select specific regions of the RG1 polypeptide for generatingantibodies. As will be understood by those skilled in the art, theregions or epitopes of an RG1 polypeptide to which an antibody isdirected may vary with the intended application. For example, antibodiesintended for use in an immunoassay for the detection of membrane-boundRG1 on prostate cells should be directed toward accessible epitopes onthe RG1 polypeptide. Regions of the RG1 polypeptide that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, or Jameson-Wolf analysis. Fragmentscontaining these residues are particularly suited in generating anti-RG1antibodies. Useful fragments include, but are not limited to, thesequences PLGGESICSAGAPAKYSIT (SEQ ID NO: 8); HSSDYSMWRKNQYVS (SEQ IDNO: 10); DAGTDSGFTFSSPNFATIPQDTV (SEQ ID NO: 11); and NEIVDSASVPET (SEQID NO: 12). Generation of polyclonal antibodies to these regions isdescribed in Example 4.

Uses of Antibodies Recognizing Epitopes of RG1

The antibodies, antigen-binding antibody fragments, and variants thereofof the invention may be particularly useful in diagnostic assays,imaging methodologies, and therapeutic methods for the management ofcancers in which RG1 is overexpressed, including cancers of theprostate, kidney, colon, and ovaries.

The invention provides various immunological assays useful for thedetection of RG1 polypeptides and for the diagnosis of cancers, such asprostate cancer. Such assays generally comprise one or more RG1antibodies capable of recognizing and binding a RG1 polypeptide. Themost preferred antibodies will selectively bind to RG1 and will not bind(or bind weakly) to non-RG1 polypeptides. The assays include variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunoabsorbent assays, and the like. In addition, immunological imagingmethods capable of detecting prostate cancer are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled RG1 antibodies. Such assays may be clinicallyuseful in the detection, monitoring and prognosis of cancers, such asprostate cancer.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Additionally, RG1 antibodies may be used to isolate RG1 positive cellsusing cell sorting and purification techniques. In particular, RG1antibodies may be used to isolate prostate cancer cells from xenografttumor tissue, from cells in culture, etc. using antibody-based cellsorting or affinity purification techniques. Other uses of the RG1antibodies of the invention include generating anti-idiotypic antibodiesthat mimic the RG1 polypeptide.

RG1 antibodies can be used for detecting the presence of prostate canceror tumor metastasis. The presence of such RG1-containing cells withinvarious biological samples, including serum, prostate and other tissuebiopsy specimens, may be detected with RG1 antibodies. In addition, RG1antibodies may be used in various imaging methodologies such asimmunoscintigraphy with a ^(99m)Tc (or other isotope) conjugatedantibody. For example, an imaging protocol similar to the one recentlydescribed using an ¹¹¹In conjugated anti-PSMA antibody may be used todetect recurrent and metastatic prostate carcinomas (Sodee et al., Clin.Nuc. Med. 21: 759-766, 1997). Another method of detection that can beused is positron emitting tomography (see Herzog et al., J. Nucl. Med.34:2222-2226, 1993).

The RG1 antibodies of the invention may be labeled with a detectablemarker or conjugated to a second molecule, such as a cytotoxic agent,and used for targeting the second molecule to a RG1 positive cell(Vitetta, E. S. et al., Immunotoxin Therapy, in DeVita, Jr, V. T. etal., eds, Cancer: Principles and Practice of Oncology, 4^(th) ed., J.B.Lippincott Co., Philadelphia, 2624-2636, 1993). Examples of cytotoxicagents include, but are not limited to ricin, doxorubicin, daunorubicin,taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D,diptheria toxin, epothilones, Pseudomonas exotoxin (PE) A, PE40, abrin,and glucocorticoid and other chemotherapeutic agents, as well asradioisotopes. Cytotoxic or antiproliferative targeted fusion proteinsmay be created by genetic or chemical fusion of the antibody to anappropriate cytokine, chemokine, interferon, or growth factor that hasthe desired anti-tumor biological activity (Asgeirsdottir et al.,Biochem. Pharmacol. 15:1729-1739, 2003). Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Suitable radioisotopes for immunotherapy or for use as adetectable marker include the following: Antimony-124. Antimony-125,Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-j206,Bismuth-207, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139,Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-56, Cobalt-57,Cobalt-58, Cobalt-60, Cobalt-64, Erbium-169, Europium-152,Gadolinium-153, Gold-195, Gold-199, Hafnium-175, Hafnium-181,Indium-111, Iodine-123, Iodine-131, Iridium-192, Iron-55, Iron-59,Krypton-85, Lead-210, Lutetium-177, Manganese-54, Mercury-197,Mercury-203, Molybdenum-99, Neodymium-147, Neptunium-237, Nickel-63,Niobium-95, Osmium-185+191, Palladium-103, Platinum-195m,Praseodymium-143, Promethium-147, Protactinium-233, Radium-2226,Rhenium-186, Rubidium-86, Ruthenium-103, Ruthenium-106, Scandium-44,Scandium-46, Selenium-75, Silver-110m, Silver-11, Sodium-22,Strontium-85, Strontium-89, Strontium-90, Sulfur-35, Tantalum-182,Technetium-99m, Tellurium-125, Tellurium-132, Thallium-170,Thallium-204, Thorium-228, Thorium-232, Tin-113, Titanium-44,Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169, Yttrium-88,Yttrium-90, Zinc-65, and Zirconium-95.

Radiolabeling of antibodies is accomplished using a chelating agentwhich is covalently attached to the antibody, with the radionuclideinserted into the chelating agent. Preferred chelating agents are setforth in Srivagtava et al. Nucl. Med. Bio. 18:589-603, 1991 and McMurryet al., J. Med. Chem. 41:3546-3549, 1998. or derived from the so-calledNOTA chelate published in H. Chong, K. et al., J. Med. Chem.45:3458-3464, 2002, all of which are incorporated herein in full byreference. Particularly preferred for conjugation of radioisotopes to anRG1 antibody are derivatives of the bifunctional chelatorp-SCN-Benzyl-DTPA (Brechbiel et al. Inorg. Chem. 25:2772-2781, 1986);for example, cyclohexyl-DTPA (CHX-A″-DTPA, Wu et al., Bioorg. Med. Chem.10:1925-1934. 1997) and MX-DTPA (1B4M-DTPA, McMurry et al., J. Med.Chem., 41: 3546-3549, 1996), as well as1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) (Chong et al. J.Med. Chem. 45:3458-3464, 2002). Conjugation can be accomplished by themethod of Nikula et al. Nucl. Med. Biol. 3:387-390, 1995. Particularlypreferred for use as a detectable marker for immunoscintigraphy are theradioisotopes ¹¹¹In or ^(99m)Tc. Preferred detectable markers forpositron emitting tomography are ⁴³Sc, ⁴⁴Sc, ⁵²Fe, ⁵⁵Co, ⁶⁸Ga, ⁶⁴Cu, ⁶⁶Yand ^(94m)Tc. For immunotherapy, the beta-emitting radioisotopes ⁴⁶Sc,⁷⁴Sc, ⁴⁸Sc, ⁷²Ga, ⁷³Ga, ⁹⁰Y, ⁶⁷Cu, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁸⁶Re, and ¹⁶⁶Re and the alpha-emitting isotopes ²¹¹At, ²¹¹Bi,²¹²Bi, ²¹³Bi and 214Bi, can be used. Preferred are ⁹⁰Y, ¹⁷⁷Lu, ⁷²Ga,¹⁵³Sm, ⁶⁷Cu and ²¹²Bi, and particularly preferred are ⁹⁰Y and ¹⁷⁷Lu.

Immunotherapy for Prostate and Other Cancers

The invention provides various immunotherapeutic methods for treatingprostate and other cancers, including antibody therapy, in vivovaccines, and ex vivo immunotherapy approaches. Other cancers includecancer of the kidney, colon, and ovaries. In one approach, the inventionprovides RG1 antibodies which may be used systemically to treat prostatecancer. For example, unconjugated RG1 antibodies may be introduced intoa patient such that the antibody binds to RG1 on, in or associated withprostate cancer cells and mediates the destruction of the cells, and thetumor, by mechanisms which may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, altering the physiologicfunction of RG1, and/or the inhibition of ligand binding or signaltransduction pathways. RG1 antibodies conjugated to toxic agents such asricin or radioisotopes, as described above, may also be usedtherapeutically to deliver the toxic agent directly to RG1-bearingprostate tumor cells and thereby destroy the tumor cells.

Prostate cancer immunotherapy using RG1 antibodies may follow theteachings generated from various approaches which have been successfullyemployed with respect to other types of cancer, including but notlimited to colon cancer (Arlen et al., Crit. Rev. Immunol. 18: 133-138,1998), multiple myeloma (Ozaki et al., Blood 90: 3179-3186, 1997;Tsunenari et al., Blood 90: 2437-2444, 1997), gastric cancer (Kasprzyket al, Cancer Res. 52: 2771-2776, 1992), B-cell lymphoma (Funakoshi etal., Immunther. Emphasis Tumor immunol. 19: 93-101, 1996), leukemia(Zhong et al., Leuk. Res. 20: 581-589, 1996), colorectal cancer (Moun etal., Cancer Res. 54: 6160-6166, 1994; Velders et al., Cancer Res.55:4398-4403, 1995), and breast cancer (Shepard et al., J. Clin.Immunol. 11: 117-127, 1991).

The invention further provides vaccines formulated to contain an RG1polypeptide or fragment thereof. The use of a tumor antigen in a vaccinefor generating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al, Int. J.Cancer 63: 231-237, 1995: Fong et al., J. Immunol. 159: 3113-3117,1997). Such methods can be readily practiced by employing an RG1polypeptide, or fragment thereof, or an RG1-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the RG1 immunogen.

For example, viral gene delivery systems may be used to deliver aRG1-encoding nucleic acid molecule. Various viral gene delivery systemswhich can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, in Curr. Opin, Immunol. 8: 658-663, 1996).Non-viral delivery systems may also be employed by using naked DNAencoding a RG1 polypeptide or fragment thereof introduced into thepatient (i.e., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human rg1 cDNA may be employed. In anotherembodiment, human rg1 cDNA fragments may be employed. In anotherembodiment, rg1 nucleic acid molecules encoding specific T lymphocyte(CTL) epitopes may be employed. CTL epitopes can be determined usingspecific algorithms (e.g., Epimer, Brown University) to identifypeptides within a RG1 polypeptide which are capable of optimally bindingto specified HLA alleles.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present a RG1 polypeptide as antigen to apatient's immune system. Dendritic cells express MHC class I and II, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., Prostate 28: 65-69, 1996; Murphy et al.,Prostate 29: 371-380, 1996). Dendritic cells can be used to present RG1polypeptides to T cells in the context of MHC class I and II molecules.In one embodiment, autologous dendritic cells are pulsed with RG1polypeptides capable of binding to MHC molecules. In another embodiment,dendritic cells are pulsed with the complete RG1 polypeptide. Yetanother embodiment involves engineering the overexpression of the rg1gene in dendritic cells using various implementing vectors known in theart, such as adenovirus (Arthur et al., Cancer Gene Ther. 4: 17-25,1997), retrovirus (Henderson et al., Cancer Res. 56: 3763-3770, 1996),lentivirus, adeno-associated virus. DNA transfection (Ribas et al.,Cancer Res. 57: 2865-2869, 1997), and tumor-derived RNA transfection(Ashley et al., J. Exp. Med. 186: 1177-1162, 1997).

Anti-idiotypic anti-RG1 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressingan RG1 polypeptide. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can be readily adapted togenerate anti-idiotypic anti-RG1 antibodies that mimic an epitope on aRG1 polypeptide (see, for example, Wagner et al., Hybridoma 16: 33-40,1997: Foon et al., J. Clin. Invest. 96: 334-342, 1995; Herlyn et al.,Cancer Immunol Immunother 43: 65-76, 1996). Such an anti-idiotypicantibody can be used in anti-idiotypic therapy as presently practicedwith other anti-idiotypic antibodies directed against tumor antigens.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing RG1. Using the RG1-encoding DNA moleculesdescribed herein, constructs comprising DNA encoding an RG1polypeptide/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take up the construct and express theencoded RG1 polypeptide/immunogen. The RG1 polypeptide/immunogen may beexpressed as a cell surface polypeptide or be secreted. Expression ofthe RG1 polypeptide/immunogen results in the generation of prophylacticor therapeutic humoral and cellular immunity against prostate cancer.Various prophylactic and therapeutic genetic immunization techniquesknown in the art may be used. (for a review, see information andreferences published at Internet address worldwideweb.genweb.com).

Assays for Identifying Agents Binding to RG1

The present invention also relates to assays and methods which can beused to identify agents (i.e. an antibody, peptide, etc.) that bind toRG1. Specifically, agents that bind to RG1 can be identified by theability of the RG1 ligand or other agent or constituent to bind to RG1and/or the ability to inhibit/stimulate RG1 activity.

As described above, antibodies are obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions, thoseportions of the RG1 polypeptide intended to be targeted by theantibodies. Such agents can be used in competitive binding studies toidentify second I generation inhibitory agents as well as to block RG1activity.

Agents that bind a RG1 polypeptide, such as a RG1 antibody, can be usedto modulate the 5 activity of RG1, to target anticancer agents toappropriate mammalian cells, or to identify agents that block theinteraction with RG1. Cells expressing RG1 can be targeted or identifiedby using an agent that binds to RG1.

How the RG1 binding agents will be used depends on the nature of the RG1binding agent. For example, a RG1 binding agent can be used to: deliverconjugated toxins, such as diphtheria toxin, cholera toxin, ricin orpseudomonas exotoxin, to a RG1 expressing cell; modulate RG1 activity;to directly kill a RG1 expressing cell; or in screens to identifycompetitive binding agents. For example, a RG1 inhibitory agent can beused to directly inhibit the growth of RG1 expressing cells whereas aRG1 binding agent can be used as a diagnostic agent.

Pharmaceutical Compositions and Administration

The present invention also relates to pharmaceutical compositions whichmay comprise rg1 polynucleotides, RG1 polypeptides, antibodies,agonists, antagonists, or inhibitors, alone or in combination with atleast one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. Any of these molecules can be administered to a patient alone, orin combination with other agents, drugs or hormones, in pharmaceuticalcompositions where it is mixed with excipient(s) or pharmaceuticallyacceptable carriers. In one embodiment of the present invention, thepharmaceutically acceptable carrier is pharmaceutically inert.

The present invention also relates to the administration ofpharmaceutical compositions. Such administration is accomplished orallyor parenterally. Methods of parenteral delivery include topical,intraarterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxilliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co,Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxilliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl, cellulose, hydroxypropylmethylcellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie. dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, andoptionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances that increase viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Kits

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Manufacture and Storage.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with may acids, including by not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in an acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of RG1, such labeling wouldinclude amount, frequency and method of administration.

Therapeutically Effective Dose.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose, i.e. treatment of aparticular disease state characterized by RG1 expression. Thedetermination of an effective dose is well within the capability ofthose skilled in the an.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., neoplastic cells, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of protein or itsantibodies, antagonists, or inhibitors that ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED₅₀/LD₅₀.Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations what include the ED₅₀, with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors that may be taken into account include theseverity of the disease state, eg, tumor size and location; age, weightand gender of the patient; diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations forpolynucleotides than for proteins or their inhibitors. Similarly,delivery of polynucleotides or polypeptides will be specific toparticular cells, conditions, locations, etc. Preferred specificactivities for a radiolabeled antibody may range from 0.1 to 10 mCi/mgof protein (Riva et al., Clin. Cancer Res. 5:3275s-3280s, 1999; Wong etal., Clin. Cancer Res. 6:3855-3863, 2000; Wagner et al., J. Nuclear Med.43:267-272, 2002).

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Example 1 Identification of Human rg1 Polynucleotide

Rg1 was identified as a gene expressed in the prostate by miningIncyte's LIFESEQ® database. The nucleotide sequence was identified by anannotation search of the database, using the “Protein Function” tootprovided by Incyte for the purpose of searching the database. Thenucleotide sequence was found in the category of cell adhesion moleculesin the annotated database and was described as a homologue of f-spondin.Electronic Northern analysis of the distribution of rg1 polynucleotidesequences in the set of libraries in the database revealed that rg1 wasexpressed at high levels in the prostate libraries and at lower levelsin a number of other tissue libraries, including those from normal andtumor tissues.

Following assembly of the set of rg1 clones in the database into acontiguous polynucleotide sequence, and editing of the contiguoussequence, a full-length coding sequence was identified in the predictedassembled polynucleotide. This sequence coded for a protein homologousto f-spondin and to Mindin-2.

Incyte clones 1640796, 1712252, and 1880265 were obtained from Incytefor experimental work and clone 3360733 was identified as containing themost 5′ nucleotide sequence. This clone was fully sequenced andcontained the full coding sequence for the predicted RG1 protein. Thissequence is shown in SEQ ID NO: 1.

Example 2 Rg1 mRNA Expression

The expression of rg1 mRNA in a variety of samples from normal and tumortissues and in cell lines, was determined by semi-quantitative PCR usinga Taqman assay, (Perkin-Elmer). Prostate normal, benign and tumor tissuesamples that had been graded according to a modified Gleason gradingsystem were obtained from the Urology Department at Stanford UniversitySchool of Medicine. RNA was isolated from these by standard procedures.RNA from other tumor and normal tissues was purchased from commercialsources, including Clonetech, and Biochain. Prostate tumor cell lines,(PC-3, LNCaP and DU145), were obtained from American Type CultureCollection and propagated in culture by standard methods using serumcontaining medium. Xenograft tumors derived from these cell lines wereestablished in nude mice and harvested from the mice approximately 4-6weeks after implantation. RNA was isolated from the tumors by standardprocedures.

Taqman based PCR analysis was performed using the primers: CGC GCA TAGCTC CGA CTA C (SEQ ID NO: 3) and GCC GCG TCC GCA MG (SEQ ID NO: 4) andthe Taqman probe: 6-FAM-AGG MG MC CAG TAC GTC AGT MC GGG CTG-Tamra (SEQID NO: 5).

These primers and probe were designed using Perkin Elmer's PrimerExpress software and were synthesized by Synthetic Genetics. PCRreactions were carried out for 30-40 cycles and quantified usingprostate RNA to generate a standard curve for relative comparison. Thisanalysis demonstrated that rg1 mRNA was detected at highest abundance inthe prostate and at significantly lower levels in several other tissues.

Example 3 Production of RG1 in BHK Cells

Cloning: The RG1 coding region was obtained from Incyte plasmid 3360733.The coding sequence was PCR amplified with primers SST115(5′-TCCCTCTAGAGCCACCATGGAAAACCCCAGCCCGGC-3′) (SEQ 10 NO: 6) and SST113(5′-AAGGCATCACGTGTTAGACGCAGTTATCAGGGACG-3′) (SEQ ID NO: 7) in a standardPCR reaction (100 ul) using 1×Pfu Turbo polymerase buffer (Stratagene,La Jolla, Calif.)/200 uM dNTPs/0.2 uM oligonucleotide primers/2.5 U PfuTurbo polymerase (Stratagene). PCR amplification conditions were asfollows: 3 mins at 95° C., (15 seconds at 95° C., 30 seconds at 60° C.,2 minutes at 72° C.)×35, 72° C. for 7 minutes. The resulting PCRamplified product was purified using a QIAquick PCR column (Qiagen,Valencia, Calif.) and digested with XbaI and PmlI restriction enzymes toresult in a 1010 by fragment that was purified from a 1% agarose gelusing a BIO 101 GeneClean Kit (Vista, Calif.). The purified fragment wasligated (using Epicientre Fast Link Kit, (Epicenter, Madison, Wis.) tothe noncytopathic Sindbis expression vector pSIN rep21 (Agapov et al,1998, PNAS 95: 12989-12994) digested with XbaI and PmlI, and transformedinto DH5 alpha competent cells (Life Technologies, Gaithersburg, Calif.)and selected on LB agar plates containing ampicillin (100 ug/ml). Onesuch ampicillin resistant colony was grown in LB medium with ampicillinand shown by sequence analysis to contain the inserted RG1 codingsequence. This plasmid was called pPEG6.

Expression: Two micrograms of pPEG6 was used to transfect 1−3×10⁵ bovinehamster kidney cells (BHK) cells using Lipofectamine Plus reagent (LifeTechnologies, Gaithersburg, Md.) according to the manufacturer'sinstructions. Following transfection, cells were incubated in DMEM plusfetal blood serum for 24-48 hours, at which time the cells were split 1to 10 and selection for the plasmid containing cells was initiated byadding puromycin (2.5 ug/ml final concentration) arid DMEM containingserum. After the cells were confluent (4-5 days post puromycin addition)the cells were washed with PBS, split 1 to 10, and DMEM medium withserum and 5 ug/ml puromycin was added. After an additional 2-3 days, themedium was replaced with DMEM and 5 ug/ml puromycin without serum, grownfor 2-3 days and the presence of RG1 protein was detected in the mediumby Western analysis using RG1 antibodies. RG1 protein was detected at alevel of 1 ug/ml.

Purification: Baby hamster kidney cells (BHK), transfected to stablyoverexpress and secrete RG1 protein into the growth medium, werecultured in medium containing fetal bovine serum. When subconfluent, thecells were switched to serum-free medium for 24-48 hours. The medium wascollected, centrifuged to remove cells and stored at −80 degrees C.Media was thawed immediately prior to purification and kept on ice.Protease inhibitors were added, media was diluted ten-fold with cold 20mM Sodium acetate buffer, pH 6.5 and kept at 4 degrees C. throughout thepurification. The diluted sample was loaded onto a Q-Sepharose anionexchange column at a flow rate of 0.5 ml/min then washed with the samebuffer. Elution was performed using a linear NaCl gradient (0-80% of 1MNaCl in buffer, 0.5% per minute) while collecting fractions. The RG1eluted at approximately 75 mM NaCl, as determined by SDS PAGE andWestern blot. RG1 containing fractions were pooled, concentrated byultrafiltration and further purified over a Superdex 75 gel filtrationcolumn. Purified BHK-RG1 was used as immunogen in the generation ofhuman mabs that are specific for native RG1 (nRG1) protein, and asantigen in the screening and characterization of these antibodies.

Example 4 Antibody Generation

Polyclonal antibodies: Rabbit polyclonal antisera were raised againstfive synthetic polypeptide sequences derived from the RG1 proteinsequence. These sequences were selected because of their predictedpositions at the surface of the protein, in order to generate antiserathat are more likely to recognize surface epitopes. Cysteine residueswere replaced with aminobutyric acid (Abu) to aid synthesis. Thespecific amino acid sequences, positions on the RG1 protein anddesignations for the five peptides are listed below.

Designation Position Amino Acid Sequence 1C 28-46 PLGGESICSAGAPAKYSIT(SEQ ID NO: 8) 2C 46-64 TFTGKWSQTAFPKQYPLFR (SEQ ID NO: 9) 3C 77-91HSSDYSMWRKNQYVS (SEQ ID NO: 10) 4C 188-210 DAGTDSGFTFSSPNFATIPQDTV (SEQID NO: 11) 5C 263-274 NEIVDSASVPET (SEQ ID NO: 12)

Peptides were covalently coupled to keyhole limpet hemocyanin (KLH), viaan additional carboxyl-terminal cysteine, for use as an immunogen.Similarly, a bovine serum albumin (BSA) conjugate was prepared for theanalysis of antisera titers via ELISA.

Two animals were immunized with each peptide. Initial immunizations wereperformed in Freunds complete adjuvant (0.5 mg/animal), followed byboosts at three week intervals with 0.25 mg/animal in Freunds incompleteadjuvant applied intramuscularly. Periodic test bleeds were taken andantibody titers against the specific BSA-peptide conjugate were measuredby ELISA and compared with preimmune sera. Antisera against peptides 1Cand 3C were shown to be active. Antisera against peptide 2C did notrecognize RG1 polypeptide. Antisera against peptides 4C and 5C were nottested.

Monoclonal antibodies: Monoclonal antibodies against RG1 were generatedby immunizing transgenic mice against RG1 peptides and a6-histidine-tagged RG1 fusion protein expressed in E. coli. Splenocytesof these animals were fused with myeloma cells to produce hybridomacells. The resulting hybridomas were screened by ELISA for hybridomasproducing antibodies directed against RG1 peptides and protein.

Human monoclonal antibodies with specificity for native RG1 were alsoprepared by immunization of transgenic mice that contain disrupted mouseheavy and mouse kappa light chain loci. (U.S. Pat. No. 5,877,397).Transgenic mice from C57BL/6J inbred strains (Medarex) were immunizedwith purified RG1 protein produced in a stable, transfected BHK cellline (see Example 3).

The antigen was mixed with Complete Freunds (Sigma, F5881) adjuvant forthe first and second immunization for protocol one; thereafter theantigens were mixed with Incomplete Freunds (Sigma, F5506). For thesecond protocol Complete Freunds was used for the first immunization andthereafter Incomplete Freunds was used. Each mouse received 25 μg nativeRG1 (nRG1) in 100 μL PBS, mixed 1:1 with the adjuvant using anemulsifying needle. Mice are injected with 0.2 mL prepared antigen intothe peritoneal cavity.

Hybridoma Preparation: The P3 X63 ag8.653 myeloma cell line (ATCC CRL1580, lot F-15183) was used for the fusions. The original ATCC vial wasthawed and expanded in culture. A seed stock of frozen vials wasprepared from this expansion. Cells are maintained in culture for 3-6months, passed twice a week. Supernatant from P388D1 (ATCC, TIB-63 FL)cells was used as conditioned media for the hybridomas. Briefly, cellswere grown and expanded to 200 ml. Stationary cultures were grown for ˜7days. The exhausted supernatant was spun down and filtered through a 0.2pm sterile filter. This cell line is passed for 3-6 months and then anew vial is thawed.

DMEM (Cellgro#10013271, 10013270) containing 5% FBS (Hyclone,#AKE11828), and PIS (Cellgro, #30002029) and were used to culture themyeloma and P388D1 cells. Additional media supplements were added to theHybridoma growth media, which included 5% Origen Hybridoma CloningFactor (IGEN, # 36684, 36908), 5% P388D1 conditioned media (Nov. 15,2000, Dec. 21, 2000 DH), 10% FCS (Hyclone, #AKE11828), β-mercaptoethanol(Gibco #1076640), Genetacin (Gibco #1079874), Hepes (Cellgro-#25060041)and HAT (Sigma, H 0262; 1.0×10⁻⁴ M Hypoxanthine, 4.0×10⁻⁷ M Aminopterin,1.6×10⁻⁵ M Thymidine), or HT (Sigma, H0137; 1.0×10⁻⁴ M Hypoxanthine,1.6×10⁻⁵ M Thymidine).

The splenocytes were fused with myeloma cells using PEG and standardmethodology. The resulting hybridomas were plated out into 50 96-wellplates, seeded at 200 μl/well for the first fusion. The initial ELISAscreen for human IgG, κ antibodies was performed 10-12 days post fusion.Human IgG, κ positive wells were then screened by a 6-His capture ELISA.This screening led to the isolation of 8 human antibodies from 3fusions: three IgM, one IgG3, and four IgG1 subclass antibodies.

Hybridomas from wells with antigen binding antibodies were firsttransferred to 24 well plates, and re-screened again for specificity,Native RG1 specific hybridomas were subcloned by limiting dilution toassure monoclonality. Hybridomas producing antibodies that bound nativeRG1 (nRG1) were preserved at several stages in the development processby freezing cells in IGEN freeze media. Media from these lines wasfrozen and used for purification of antibodies as described below. Fourof the eight were determined to have sufficient specificity to warrantfurther study.

Purification of antibodies: Four of the human monoclonal antibodies toRGI described above were purified from cell conditioned media usingProtein G Sepharose affinity chromatography. Cells were removed frommedia by centrifugation and filtration, the media was passed over theProtein G column. The column was then washed in PBS to remove unboundmaterial. Bound antibodies were eluted with 100 mM glycine, pH 2.5 andimmediately neutralized by adding 10% v/v 1M Tris, pH 8 to thefractions. Fractions containing antibody were pooled, dialysed into PBS,tested for purity by SDS PAGE, and assayed for antigen binding activityby ELISA.

Screening of Antibodies: Screening of antibodies was performed usingseveral different assay procedures:

A. hlgGγκ ELISA Screen: Ninety-six well microtiter plates (Falcon,#3912) were coated overnight with 1 ug/ml anti-human IgGκ or anti-humanIgGκ in PBS (50 ul/well). Plates were aspirated and blocked with PBS0.05% Tween 20 containing 5% chicken serum for 1 hour at roomtemperature (100 μl/well) then washed three times with PBS-tween.Hybridoma supernatants were diluted 1:2 in blocking buffer and incubatedfor 1 hour at room temperature (100 μl/well) for screening. Followingincubation, the plates were washed three times in blocking buffer priorto adding 100 μl/well of secondary antibody (HRP anti-human IgGFc(Jackson, #109-036-098 or HRP anti-human IgGκ (Sigma, #A-7164). Thesecondary antibody was incubated for 1 hour at room temperature and thenthe plates were washed 2× in blocking buffer. Plates were developedusing 10 ml citrate phosphate buffer, pH 4.0 containing 80 μl ABTS(Sigma, #A1888), 8 μl H₂O₂ per plate and read at A₄₁₅₋₄₉₀ nm.

B. RG1 Binding ELISA: Ninety-six well microtiter plates were coatedovernight with 0.5-1.0 ug/well purified native RG1 protein in PBS, 50ul/well, 4 degrees C. The wells were aspirated and then the reaction wasblocked with the addition of 100 ul/well PBS-tween+5% chicken serum,followed by incubation for 1 hour at room temperature. Plates were thenwashed 3 times in blocking buffer. Serially diluted samples (serum,hybridoma supe, purified mabs etc.) were then added to each well, at 50ul/well. Incubate 1 hour at room temperature then wash 3 times inblocking buffer. The wells are then incubated with HRP anti-human IgGFcsecondary antibody in blocking buffer for 1 hour at room temperature andthen washed 3 times as before. Plates are developed using substratedescribed above and measurement of the A₄₀₅ nm using a 96 well platereader.

C. Capture ELISA Method: In order to determine mab binding to RG1protein in its native conformation, a capture ELISA format was used. RG1protein, containing a six histidine expression tag (6His-RGI), wasexpressed in BHK cells and used as the antigen. The 6His-RG1 waspurified from conditioned media using NiNTA agarose affinitychromatography following standard methodology.

Purified 6H is RG1 was captured on 96 well NiNTA plates (Qiagen NiNTAHisSorb) using a concentration of 1.5 ug/ml in PBS plus 0.2% BSA(PBS/BSA, 100 μl/well) overnight at 4 degrees C. Wells were washed 3times with PBS containing 0.05% Tween 20 (PBST). Samples (hybridomasupernatant, sera, purified mabs etc.) were diluted in PBS/BSA andincubated on plates for 1-2 hours at room temperature (50 μl/well) andthen washed 3 times with PBST. Secondary antibody (HRP-labeled goatanti-human IgGFc) was diluted 1:5000 in PBS/BSA, added to the plate at50 ul/well and incubated for 1 hour at room temperature.

Plates were washed 3 times with PBST and developed as in an ELISA.Absorbance at 405 nm was measured using an ELISA plate reader.

D. BIAcore Surface Plasmon Resonance (SPR) Assay: Parental hybridomasupernatants were further screened to qualitatively rank clones byavidity using SPR. A rabbit anti-human IgGFc (Pierce, 31142) wasimmobilized onto the sensor chip (Biacore, BR-1000-12) using standardamine coupling and 60 ug/ml antibody in acetate pH 4.0 and a mobilephase of HEPES buffered saline (HBS). Hybridoma media was passed overthe surface at 5 ul/min to capture onto the surface and then washed tobaseline with HBS. Purified, native, BHK-RG1 protein (400 nM) was thenpassed over the surface and binding was measured by SPR. At the end ofthe injection, HBS was passed over the surface to measure dissociationof the antibody:RG1 complex. The slope of the SPR measurement over timeis indicative of the dissociation rate, the greater the slope, thefaster the off rate and therefore the lower avidity of the antibody.

Example 5 Western Blot Analysis of Antibodies

Antisera were tested for RG1 specificity via Western blotting. RG1specific antisera (those raised against sequences 1C and 3C, above) weretested on RG1 transiently expressed in COS cells, native RG1 secretedfrom LNCaP cells and RG1 produced from transfected baby hamster kidneycells (BHK). RG1-specific antisera were further tested on lysatesprepared from: LNCaP tumors, LNCaP cells, PC3 tumors, PC3 cells andseveral clinical samples of human prostate tumors. Cells and tissueswere lysed in detergent buffer. After boiling for 5 min, 10 ul of eachlysate was loaded onto a 12% SDS-polyacrylamide gel to resolve proteins.Separated proteins were then transferred to nitrocellulose membranes.Binding specificity of RG1 antibodies was verified by binding in thepresence of the homologous and heterologous peptides. RG1-specificantisera could detect the protein in all samples but PC-3 cells and PC-3tumors.

Western blot analysis of human mabs specific for native RG1 demonstratedthat these antibodies recognized RG1 on blots only under non-reducingconditions. This suggested that these mabs bind to a more native form ofRG1.

Example 6 Purification of Native RG1 Protein Secreted from LNCaP Cells

LNCaP cells grown in culture were shown to secrete native RG1 protein byWestern blot analysis. In order to purify the native protein, cells weregrown for 48 hours in media lacking serum. This serum-free conditionedmedia was harvested, centrifuged to remove any cells, and concentratedapproximately fifty-fold by ultrafiltration. The concentrated media wasthen diluted ten-fold with 20 mM sodium acetate buffer, pH 6.5 andloaded onto a Q-Sepharose anion exchange column. Column elutionconsisted of a sodium chloride gradient (0.5% per minute) whilecollecting 2.0 ml fractions. The RG1 protein eluted at approximately 75mM NaCl as determined by Western blot and SDS PAGE. The native RG1protein runs at a slightly lower molecular weight than the 6histidine-RG1 fusion protein expressed in bacteria, presumably becauseit lacks the fusion peptide.

Example 7 Immunohistochemical staining of RG1 Expression in Prostate andProstate Cancer Metastasis

The expression of RG1 protein was determined by LifeSpan Biosciences,Inc. in a variety of human tissues, including kidney, lung, pancreas,muscle, brain and prostate, as well as lymph node and bonemetastasis.Additional prostate tissues were obtained from the Urology Department atStanford University School of Stanford and tested at Berlex. The tissuesections were deparaffinized using standard procedures. The polyclonalantibody RG1-3C was used as a primary antibody and the detection systemconsisted of using Vector ABC-AP kit (AK5002) with a Vector redsubstrate kit (Sk5002). As a negative control, the staining was carriedout in the absence of the primary antibody.

Expression of RG1 was examined in prostate tumor and normal prostatetissue from several patients. In all cases, strong staining,representing RG-1 expression, was seen in the prostate tumor samples.RG-1 expression varied in the normal prostate tissue, from almost noneto significant staining. Expression of RG1 was also detected byimmunohistochemistry in lymph node and bone samples known to containprostate tumor metastasis. Normal lymph node or bone do not showstaining.

Example 8 Sequencing of RG1 Antibodies

The nucleic acid sequences of two human RG1 antibodies (C and B)generated and purified as described above in Example 4 were determinedby standard methods. The nucleotide sequence of the B light chainvariable region is designated SEQ ID NO: 20 and that of the B heavychain variable region is SEQ ID NO: 21. The nucleotide sequence of the Clight chain variable region is designated SEQ ID NO: 23 and that of theC heavy chain variable region is SEQ ID NO: 24.

The corresponding predicted amino acid sequences of these variable chainregions were determined, and are designated SEQ ID NO: 26 (B lightchain); SEQ ID NO: 27 (3 heavy chain); SEQ ID NO: 29 (C light chain);SEQ ID NO: 30 (C heavy chain). See FIGS. 3 and 4.

Example 9 Determination of Binding Constants for RG1 Antibodies

Kinetic constants (K_(D), k_(a) and k_(d)) of mab binding to native RG1protein were determined by BIAcore using a capture format in whichsoluble, native RG1 protein was bound to immobilized mab on a sensorchip. ImmunoPure rabbit anti-human IgGFc (Pierce, 31142) was covalentlyimmobilized to the Sensor Chip CM5 (Biacore, BR-1000-12) using standardamine coupling methods. 100 ug/ml antibody diluted in 10 mm acetate, pH4.0 was used at 5 ul/min. HBS-EP (Biacore, BR-1001-88) was used asmobile phase. Unreacted sites were blocked with ethanolamine. Mabs werediluted to 200 nM with HBS and 50 μl was injected per cycle at 10ul/min. Serial dilutions of BHK-RG1 (12.5-400 nM) were bound to theimmobilized mab. Dissociation kinetics were measured immediately afterthe antigen injection was completed at 20 μl/min for 8 minutes. Thesurface was regenerated following each cycle using 25 ul of 10 mmglycine, pH 1.8 and then washed with HBS.

Typically, five concentrations and a media control were run. Data werefit to a 1:1 Langmuir model using the software provided by theinstrument manufacturer (BIAevaluation 3.0), and kinetic constants werecalculated. Equilibrium constants were in the nanomolar range withfavorable dissociation rates (10⁻⁴ s⁻¹). Table 1 shows the kineticconstants for 4 of the human antibodies.

TABLE 1 Kinetic constants of human RG-1 antibodies A, B, C, and D. Thekinetic constants for these antibodies were determined by fitting a 1:1Langmuir model to the data obtained from the BIAcore study. M ab K_(a)(1/M s) K_(d) (1/s) k_(D) (M) A 2.3 × 10⁴ 1.9 × 10⁻⁴  8.9 × 10⁻⁹ B 2.9 ×10⁴ 2.3 × 10⁻⁴  8.4 × 10⁻⁹ C 2.5 × 10⁴ 8.4 × 10⁻⁴ 3.36 × 10⁻⁸ D 3.17 ×10⁴  2.95 × 10⁻³  9.27 × 10⁻⁸ K_(a): association rate (1/s); k_(d):dissociation rate (1/s) K_(D): affinity (M)

Example 10 Radiolabeling of RG1 Antibodies

Conjugation of chelator to RG1 antibodies: The bifunctional chelatorp-SCN-Benzyl-DTPA (Macrocyclics, Inc.) was covalently attached toantibodies using a method adapted from Nikula et al, Nucl. Med. Biol.,Vol. 22, No. 3, pp. 387-390, 1995. All reagents and equipment utilizedduring this procedure were rendered metal-free prior to use in order toavoid inactivation of the chelator. All solutions were prepared with lowmetal reagents, high purity (MilliQ) water and Chelex treated to removetrace metals, All equipment was rinsed with 10 mM EDTA and thenextensively rinsed with MilliQ water.

Purified mabs (˜20 mg) were first treated with 1 mM EDTA for 1 hour atroom temperature to remove any metals prior to buffer exchange into 50mM Carbonate buffer, 150 mM NaCl, pH 8.5 using a Pharmacia 26/10desalting column on an AKTA chromatography system. Antibody containingfractions were pooled and concentrated to approximately 2 mg/ml byultrafiltration (Centricon 30). A 100 mg/ml p-SCN-Benzyl-DTPA stocksolution was freshly prepared in anhydrous DMSO. A 50-100 fold molarexcess of DTPA was used in the conjugation reaction, which was allowedto proceed overnight at room temperature. The reaction mixture was thenbuffer exchanged into 50 mM Na Acetate, 150 mM NaCl, pH 6.5(Radiolabeling Buffer) and concentrated to at least 3 mg/ml byultrafiltration. Antibody conjugates were stable in this buffer forweeks at 4 degrees C. Protein concentration was determined by BCA andantigen binding activity was verified by ELISA.

Radiolabeling of RG1 antibodies: DTPA-conjugated antibodies wereradiolabeled with ⁹⁰Y or ¹¹¹In, under metal-free conditions, for use inin vivo studies in tumor-bearing animals. Typically, 10 mg of antibodyconjugate was mixed with 10 mCi of [⁹⁰Y]Cl₃ or [¹¹¹In]Cl₃ (PerkinElmerLife Sciences) for 1 hour at room temperature with gentle mixing behindheavy shielding. EDTA was added to 1 mM and incubated for 10 minutes atroom temperature. The reaction mixture was run over a Pharmacia 26/10Desalting column, that had been preequilibrated in metal-free PBS, inorder to separate the radiolabeled antibody from unbound ⁹⁰Y and toexchange buffer. One ml fractions were collected and antibody containingfractions were pooled. Protein concentration was determined by BCA.Total radioactivity in a 1 ul sample was determined using a liquidscintillation counter for ⁹⁰Y or a gamma counter for ¹¹¹In. Specificactivity was calculated as mCi per mg of protein, and typically rangedfrom 0.25 to 1.0 mCi/mg. Radiological purity was determined by instantthin layer chromatograph (ITLC) according to Nikula et al, Nucl. Med.Biol. 22: 387-390, 1995. Typically, greater than 98% of theradioactivity was associated with the protein. Antigen binding activityof the radioconjugate was determined by ELISA against an unconjugatedantibody standard (⁹⁰Y conjugates), or using a solid phase radioimmunebinding assay on immobilized RG1 protein (¹¹¹In conjugates). In allcases, antigen binding of the radioconjugates was indistinguishable fromthat of the unconjugated antibody.

Example 11 Tumor Specific Accumulation of ¹¹¹In-Labeled RG1 Antibodies

Tumor xenografts were established by s.c. injection of 1×10⁻⁷ LNCaPcells in matrigel into the flank of 5-6 week-old male athymic nude mice.Biodistribution studies were performed when the tumors reached a volumeof 150-400 mm³ (approximately 4-6 weeks after tumor cell inoculation).¹¹¹In-labeled human RG1 antibodies (C, B, A) and a non-specific humanIgG₁ control antibody (specific activities, 0.3 mCi/mg) wereadministered intravenously into four groups of 12 mice bearing LNCaPxenografts. Mice were exsanguinated by cardiac puncture prior todissection. Blood, tumor and all major organs were removed, weighed onan analytical balance, and the radioactivity was counted in agamma-counter. The whole body clearance was determined by summing theradioactivity measured in blood, individual organs, and in the remainingcarcass. All data were corrected for radioisotope decay. Results wereexpressed as percentage injected dose per gram of tissue. RG1 specificantibodies show a high tumor specific accumulation (See FIG. 1).

Example 12 Tumor Growth Inhibition with ⁹⁰Y-Labeled RG1 Antibodies

Tumor xenografts were established by s.c. co-inoculation of 1×10⁻⁷ LNCaPcells in matrigel into the flank of 5-6 week-old male athymic mice.Treatment was initiated when the tumors reached a volume of 50-350 mm³(5 weeks after tumor cell inoculation). Tumor bearing animals wereevenly distributed into four treatment groups (n=13/group). A singlei.p. injection of radiolabeled antibody B, C, and non-specific IgG₁ (125μCi/animal) was administered into mice bearing LNCaP xenografts. Thefourth treatment group was given a saline i.p. injection. The effect of⁹⁰Y labeled RG1 specific antibodies on the growth of the LNCaP-derivedtumors was monitored for 32 days after injection. At that time, animalswere sacrificed and tumors were taken out and weighed. Health status wasdetermined by monitoring body weight. A single administration of ⁹⁰Ylabeled specific human RG1 antibodies produced a significant inhibitionof tumor growth when compared to the results seen in the animalsinjected with ⁹⁰Y labeled nonspecific antibody or the vehicle control.(See FIG. 2).

Example 13 Cloning and Expression of RG1 Antibodies in CHO Cells

Mutagenesis: Site-directed mutagenesis of the wild-type cDNA encodingvariable regions of anti-RG1 antibody B and C was carried out togenerate allotypes that are more frequently expressed in humans.Multisite-directed mutagenesis was performed using a kit sold byStratagene (QUICKCHANGE®) to conduct the mutagenesis, with TOPO/BVH andTOPO/CVH (Medarex) as templates. Primers(GGGGAGGCTTGGTACAACCTGGGGGGTCCCTGAG: SEQ ID NO: 14) and(GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAG; SEQ ID NO: 15) were usedto introduce the point mutations H13Q, M90T and MB2V into B cDNA(BVH_(—)3m); and H13Q, M90T into C cDNA (CVH_(—)2m). Mutations wereconfirmed by DNA sequence analysis and resulted in the mutant heavychain variable regions with sequences of SEQ ID NO: 22 and SEQ ID NO:25, respectively. The predicted amino acid sequences for these two heavychain variable regions are given by SEQ ID NOS: 28 and 31, respectively.

Construction of expression vectors: The expression vector of pIE_SRγ1fa(Medarex) contains cDNAs encoding CH and CL regions of human IgG1 (fahaplotype) and kappa chains, respectively. To allow for in frame cloningof B and C light chain variable regions into pIE_SRγ1fa was the primerpair BVK_F (GGGAAGCTTGCCACCATGGAA ACCCCAGCG; SEQ ID NO: 16) and BVK R(CAGTCGTACGTTT GATCTCCACCTTGGTCC; SEQ ID NO: 17) was used to introducecompatible HindIII/Bsiw sites (under line) at the 5′ and 3′ ends,respectively, of BVL and CVL cDNA. The resulting PCR-generated VL cDNAswere cloned into the HindIII/Bsiw site of pIE-SRγ1fa to create pIE/BVLand CVL. The same strategy was used for constructing in frame VH fusions(including BVH, BVH_(—)3m, CVH and CVH_(—)2m) into pIE/BVL and CVL.Briefly, the primer pair of CVH_F(GTCAGGATGCGGCCGCCACCATGGAGTTGTGCTGAGCT; SEQ ID NO: 18) and CVH_R(ACCGATGGGCCCTTGGTGGA; SEQ ID NO: 19) was used to introduce NotI/ApaIsites at the ends of PCR-amplified VH cDNA. The PCR products weredigested with NotI/ApaI and inserted upstream of the CH region ofpIE/BVL and pIE/CVL ensuring that the VH regions were in frame with CHregion in the respective pIE derivatives. The final constructs werenamed pIE/B, pIE/B_(—)3m, pIE/C and pIE/C_(—)2m. All inserts have beenverified by DNA sequence analysis.

Transfection and selection/amplification of DG44 and DXB11 cells. About4×10⁶ DG44 and DXB11 cells supplemented with F12 medium and 5% FCS wereplated on P100 dishes one day before transfection. Transfections werecarried out using Lipfectamine 2000 (Invitrogen) and 24 μg linearizedplasmid DNA (pIE/B_(—)3m or pIE/C_(—)2m)/P100. The medium was changed 4hours after transfection. Selective conditions were appliedapproximately 24 h post-transfection.

Selection was first carried out with MEM medium containing 5% dialyzedFBS, 2 mM L-glutamine and G418 (400 ug/ml), but lacking ribonucleosidesand deoxyribonucleosides. Reaching a confluency of about 90% cells weresplit into 4×P100 dishes and co-selected with G418 plus methotrexate atvarious concentrations. After one week, the surviving cells were platedinto 96-well plates at 100-cells/plate in the presence of co-selectionmedium. Surviving clones were screened by ELISA for expression ofrecombinant antibody. Gene copy number of 10 clones exhibiting thehighest expression levels was amplified by consecutive selection in thepresence of increasing concentrations of methotrexate and chosen clonesadapted to serum free medium for preparation of a master cell bank.

All publications and patents mentioned in the above specification areherein incorporated by reference. While the present invention has beendescribed with reference to the specific embodiments thereof, it shouldbe understood by those skilled in the art that various changes may bemade and equivalents may be substituted without departing from the truespirit and scope of the invention. In addition, many modifications maybe made to adapt a particular situation, material, composition ofmatter, process, process step or steps, to the objective, spirit andscope of the present invention. All such modifications are intended tobe within the scope of the claims appended hereto.

1. An isolated human antibody, or antigen-binding antibody fragmentthereof, or a variant thereof, that specifically binds to an epitopepresent in an RG1 polypeptide. 2-36. (canceled)
 37. An isolated human orhumanized antibody or an antigen-binding fragment thereof, which: (i)binds RG1 polypeptide having the amino acid sequence of SEQ ID NO:2, and(ii) competes for the same epitope as the epitope bound by the antibodyselected from the group consisting of: a) an isolated antibody or afragment thereof comprising a light chain variable region comprising theamino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 29, or b) theisolated antibody or a fragment thereof comprising a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 27, SEQID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 31 or c) an isolated antibody ora fragment thereof comprising a light chain variable region having theamino acid sequence of SEQ ID NO: 26 and a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, or d)an isolated antibody or a fragment thereof comprising a light chainvariable region having the amino acid sequence of SEQ ID NO: 29 and aheavy chain variable region having the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:
 31. 38. An isolated human or humanized antibody or anantigen-binding fragment thereof, which: (i) binds RG1 polypeptidehaving the amino acid sequence of SEQ ID NO:2, and (ii) competes for thesame epitope as the epitope bound by the antibody comprising: a) a lightchain CDR3 sequence comprising the amino acid residues 110 to 117 of SEQID NO: 26, b) a heavy chain CDR3 sequence comprising the amino acidresidues 117 to 132 of SEQ ID NO: 27, c) a light chain CDR1 sequence andCDR2 sequence, wherein the sequences comprise the amino acid residues 44to 55 of SEQ ID NO: 26 and 71 to 77 of SEQ ID NO: 26, respectively, andd) a heavy chain CDR1 sequence and CDR2 sequence, wherein the sequencescomprise the amino acid residues 50 to 54 of SEQ ID NO: 27 and 69 to 84of SEQ ID NO: 27, respectively.
 39. An isolated human or humanizedantibody or an antigen-binding fragment thereof, which: (i) binds RG1polypeptide having the amino acid sequence of SEQ ID NO:2, and (ii)competes for the same epitope as the epitope bound by the antibodycomprising: a) a light chain CDR3 sequence comprising the amino acidresidues 110 to 117 of SEQ ID NO: 29, b) a heavy chain CDR3 sequencecomprising the amino acid residues 117 to 126 of SEQ ID NO: 30, c) alight chain CDR1 sequence and CDR2 sequence, wherein the sequencescomprise the amino acid residues 44 to 55 of SEQ ID NO: 29 and 71 to 77of SEQ JD NO: 29, respectively, and d) a heavy chain CDR1 sequence andCDR2 sequence, wherein the sequences comprise the amino acid residues 50to 54 of SEQ ID NO: 30 and 69 to 84 of SEQ ID NO: 30, respectively. 40.The antibody fragment of any one of claim 37, 38 or 39, wherein theantibody fragment is selected from a group of fragments consisting ofFv, F(ab′), F(ab′)₂, scFv, minibody and diabody fragments.
 41. Theantibody fragment of any one of claim 37, 38 or 39, wherein the antibodyis a monoclonal antibody.